1. Field of the Invention
The present invention relates generally to the field of thrombosis. The invention particularly provides zebrafish screening methods for use in identifying anti-thrombotic agents for therapeutic use and for use in identifying genes associated with all aspects of thrombus formation, including in humans. The preferred methods of the invention involve laser irradiation injury, sodium hydroxide-induced gill bleeding and red cell lysis assays conducted in zebrafish.
2. Description of Related Art
Thrombosis is a leading cause of death in the western world. According to Virchow, thrombosis arises from disturbances to three components: the vessel wall, the constituents of blood or the blood flow—classically known as Virchow's triad (Virchow, 1856). Recent studies on the components of Virchow's triad have shown that thrombosis is a multifactorial disease, involving the interaction of numerous factors leading to the formation of a thrombus, which may result in myocardial infarction, ischemia, and stroke (Zoller et al., 1999). In light of this multifactorial nature, it has been difficult to predict the occurrence of thrombotic attack.
Extensive genetic analysis of individuals with a family history of thrombosis has resulted in the identification of a limited number of genetic mutations and polymorphisms associated with an increased risk of thrombosis (Schafer, 1994). For example, genetic studies in humans identified factor V Leiden mutation and prothrombin 3′-end polymorphism as two of the more common mutations associated with increased risk of venous thrombosis (Reitsma, 2001). However, this analysis has not proven capable of identifying genetic risk factors in a significant number of the cases of inherited venous thrombosis (Reitsma, 2001; Robetorye & Rodgers, 2001). Even less is known about major genetic risk factors in arterial thrombosis (Williams & Bray, 2001). Additionally, the interaction between enviromnental factors and polymorphisms in hemostatic factors that lead to thrombus formation is poorly understood (Schafer, 1994).
A complete understanding of the various elements in Virchow's triad that contribute to thrombosis is clearly lacking. Further insight into additional genetic factors affecting thrombosis, as well as an increased knowledge of in vivo thrombus formation, is therefore of paramount importance in improving human health.
In patients that suffer from a thrombotic attack, anticoagulant therapy is necessary. Heparin is currently the clinical anticoagulant drug of choice, and is used universally for prophylaxis of postoperative thromboembolism, in patients with stroke, during various surgical situations, and in procedures involving extracorporeal blood circulation. Extracorporeal blood circulation is employed in numerous clinical situations, such as kidney dialysis, open-heart operations, cardiac catheterizations, blood oxygenation, plasmapheresis, organ transplantation, and the implantation of artificial organs.
Systemic heparinization, however, has been reported to result in a high incidence of bleeding complications, with major bleeding occurring in 8% to 33% of patients who receive various forms of heparin therapy. This is one line of evidence highlighting the need for new anti-thrombotic agents for therapeutic use. The development of new therapeutic agents first requires in vitro and in vivo screening methods of sufficient predictive value to permit pre-clinical testing. Reliable screening methods are lacking in the field of thrombosis.
Two of the strategies that can be employed to identify additional genetic factors in thrombosis are, first, more extensive screening of human populations prone to thrombosis using recently available genomic information; and second, global genetic screens for thrombosis in biochemical and animal models. Although more extensive genetic screening in disease-prone human populations is useful, these analyses cannot be applied to the development of new pharmaceutical products to treat diseases and disorders associated with aberrant thrombosis.
Biochemical, cellular and animal screening assays useful in research in other systems, such as those relying on yeast or drosophila, are evidently unsuitable to studies of thrombosis, which requires intact hemostatic pathways. The mouse has been a popular model in genetic studies of disease, due to the availability of knockout technology. Knockout models of certain hemostatic factors have been generated in the mouse (Hogan et al., 2002). However, only a limited number of hypercoagulable states were produced in these genetic studies, such as the disruption of protein C (Jalbert et al., 1988) and protein Z (Yin et al., 2000).
Genetic studies of thrombosis in the mouse model are limited by the labor intensity of generating knockouts. In addition, the generation of null alleles may result in embryonic lethality, which yields no relevant information on thrombosis. Although mutagenesis techniques could potentially overcome this latter problem, by generating point mutations that may cause hypo- and hyperactive alleles in addition to null alleles, large-scale mutagenesis screens for identifing novel genes affecting thrombosis are unfortunately lacking in the mouse model.
Accordingly, there remains in the art a need for improved screening assays related to the study of thrombosis, particularly those that can be applied to the identification of risk factors indicative of thrombosis and to the development of anti-thrombotic agents for use in treatment. The development of assays in an animal model in which the hemostatic pathways are similar to those of humans and yet which are amenable to large scale screening is particularly desirable.